Faculty Bibliography

Background Chimeric antigen receptor (CAR) T-cell therapy has shown clinical efficacy in hematologic cancers, but success is limited in solid tumors due to a lack of tumor-specific targets that distinguish cancer from normal cells and an immunosuppressive tumor microenvironment.1 Integrating synthetic biology and comprehensive molecular profiling of tumors may provide active and tolerable approaches to CAR T-cell therapy in patients with solid tumors. Human leukocyte antigen (HLA) loss of heterozygosity (LOH) in tumors offers a definitive tumor vs normal discriminator target for CAR T-cell therapy.2 The Tmod platform3,4 is a modular logic-gated CAR T system comprising different versions including a carcinoembryonic antigen (CEA)- or mesothelin (MSLN)-targeting CAR activator and a separate blocker receptor targeting HLA-A*02 or other HLA alleles to protect normal cells. Compared with existing immunohistochemistry (IHC) tests, Tempus xT-Onco is a standard-of-care next-generation sequencing (NGS) assay5 that detects somatic alterations including HLA LOH and generates whole transcriptome RNA data (eg, CEA or MSLN expression) and a tumor immune infiltration profile, which can effectively identify patients appropriate for Tmod CAR T-cell therapy. Methods HLA LOH in solid tumors was assessed with paired germline and somatic DNA sequencing. Common driver mutations, microsatellite instability status, and tumor mutational burden were examined in HLA-A LOH or HLA-A intact cohorts. Tumor expression of CEA and MSLN was evaluated via RNA sequencing and compared with immunohistochemistry (IHC) results. Results A total of 21,053 tumor samples in the Tempus database were compared with their matched-normal samples. HLA-A LOH was detected in 16% of 10,867 advanced solid tumors (table 1) and similar LOH frequencies were observed among common HLA-A alleles. Clinical factors and molecular biomarkers were similar between HLA-A LOH and HLA-A intact cohorts. High CEA expression was seen in IHC-positive patients. Conclusions The frequency of HLA-A LOH in solid tumors in the Tempus database is similar to that reported in the Cancer Genome Atlas.6 Tempus xT-Onco reliably detects HLA LOH and quantifies CEA and MSLN expression. Based on these data, patients with solid tumors are now being prospectively screened for HLA LOH using xT-Onco in an ongoing tissue banking study (BASECAMP-1, NCT04981119), preparing for future interventional protocols

Background Nearly all colorectal and most pancreatic and lung cancers express carcinoembryonic antigen (CEA). However, due to its expression in normal gut epithelial cells, CEAtargeted therapies have resulted in on-target, off-tumor toxicity. To overcome this, we have developed TmodTM, a logicgated T-cell therapy platform. Tmod constructs are composed of an activating CAR or T-cell receptor that targets a tumor antigen and an inhibitory receptor recognizing an antigen expressed on normal healthy tissues, but not on tumor cells due to loss of heterozygosity (LOH).1,2 A2B530 is a CEAdirected Tmod construct utilizing an LIR-1-based inhibitory receptor (blocker) targeting human leukocyte antigen A*02 (HLA-A*02). Methods To generate CEA Tmod, T cells from HLA-A*02(+) donors were transduced with a single lentivirus to express i) the CAR, ii) the blocker, and iii) an shRNA targeting b2M. Cytotoxicity was measured by culturing CEA(+) target cell line pairs (A*02[-] and A*02[+]), expressing either GFP or RFP, with engineered T cells and quantifying live target cells over time. In vivo activity was examined using NSG mice subcutaneously implanted with normal (CEA[+]A*02[+]) and tumor cells (CEA[+]A*02[-]), in the right and left flanks. Mice were treated intravenously with CEA Tmod cells or control T cells. Results Control CEA CAR T cells killed CEA(+) target cell lines in vitro irrespective of HLA-A*02 expression. In contrast, CEA Tmod cells selectively killed tumor cells (CEA[+]A*02[-]) while sparing normal cells (CEA[+]A*02[+]). In mixed target cell cultures, CEA Tmod cells killed only the A*02(-) target cells, whereas the CEA CAR T cells killed both the A*02(- ) and A*02(+) cell lines. Further, CEA Tmod cells exhibited bidirectional control between the activated and blocked states. While mice treated with control CEA CAR T cells experienced a reduction in volume and bioluminescence of both normal and tumor grafts, CEA Tmod cells specifically cleared A*02(-) tumors in mice (table 1). Finally, although expansion of Tmod cells in peripheral blood trended lower than CAR and TCR controls, anti-tumor activity was comparable in these groups. Conclusions A2B530 is an autologous CEA Tmod cell product that exploits common LOH at the HLA locus in cancer cells, enabling these engineered T cells to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study identifying patients with somatic HLA LOH, is recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B530 EVEREST-1 interventional study

Background Mesothelin (MSLN) is expressed on a variety of solid tumors, including mesothelioma and ovarian, uterine, gastric, pancreatic, and lung cancers.1 However, efforts to target MSLN using cellular therapies have been hampered by severe on-target, off-tumor toxicities associated with damage to normal tissues expressing MSLN.2 To avoid these toxicities, we have developed a logic-gated engineered cell therapy, TmodTM, which is composed of two chimeric antigen receptors (CARs): an activator that targets a tumor-associated antigen and an inhibitory receptor (blocker) gated by an antigen expressed on normal tissue but lost in tumor cells due to loss of heterozygosity (LOH). A2B694 is an MSLN-specific Tmod construct combining a third-generation MSLN CAR with an LIR-1-based inhibitory receptor specific for human leukocyte antigen A*02 (HLA-A*02). Methods Lentivirus encoding i) the CAR, ii) the blocker, and iii) an shRNA targeting b2M was used to transduce T cells from HLA-A*02 donors and generate MSLN Tmod cells. In vitro cytotoxicity measurements were performed using fluorescence-based imaging and luciferase readouts. In vivo assessments were performed in NSG mice subcutaneously implanted with normal cells (MSLN[+]A*02[+]), or tumor cells (MSLN[+]A*02[-]), in the left and right flanks, respectively. Following engraftment, mice were randomized and treated intravenously with MSLN Tmod cells or controls. Grafts were measured via caliper. Results MSLN Tmod cells preferentially killed tumor cells (MSLN[+]A*02[-]) over normal cells (MSLN[+]A*02[+]) in vitro, unlike clinically active comparator M5 CAR T cells, which indiscriminately killed both target cell types (figure 1A). Soluble MSLN, tested across a 0-2 mg/mL range, did not impact MSLN Tmod function. Additionally, in mixed cell cultures where T cells and tumor and normal cells were simultaneously cultured (1:1:1 ratio), MSLN Tmod cells selectively killed tumor targets while sparing normal cells. Further, MSLN Tmod cells cycled between activated and blocked states in vitro when repeatedly challenged with tumor or normal target cells. Finally, while MSLN CAR T cells killed both normal and tumor grafts in vivo, MSLN Tmod cells selectively killed tumor grafts while sparing normal grafts (figure 1B, C). Conclusions A2B694 is an autologous MSLN Tmod cell product that leverages LOH at the HLA locus in cancer cells, providing a mechanism to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study that will identify patients with somatic HLA LOH, is currently recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B694 interventional study (EVEREST-2)

Background Solid tumors comprise >90% of cancers. Nonsmall cell lung cancer (NSCLC), metastatic colorectal cancer (CRC), and pancreatic cancer are the leading causes of cancerrelated mortality (5-year overall survival: 26%, 15%, and 11%, respectively).1 Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies.2,3 However, translating engineered T-cell therapies to solid tumors has proven to be challenging due to a lack of tumor-specific targets that can discriminate cancer cells from normal cells. Previous studies using carcinoembryonic antigen (CEA) T-cell receptors and mesothelin (MSLN) CARs resulted in dose-limiting on-target, off-tumor toxicities.4,5 To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust logic gate capable of killing tumor cells, while leaving healthy cells intact.6,7 The 2 receptors in Tmod CAR T-cell therapy comprise an activator that recognizes an antigen on the surface of tumor cells that may also be present on normal cells, such as CEA and MSLN, and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells (figure 1).8,9 Human leukocyte antigen (HLA) loss of heterozygosity (LOH) offers a definitive tumor versus normal discriminator target for CAR T-cell therapy.10 The frequency of HLA LOH among advanced NSCLC, CRC, and pancreatic cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%- 23.1%.11 LOH can be reliably detected using the Tempus xTOnco next-generation sequencing (NGS) assay.12,13 Different activator/blocker combinations can be engineered with the Tmod platform technology and may be applied to T cells and natural killer cells in autologous and allogeneic settings. BASECAMP-1 is a currently enrolling observational study with key objectives: 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) Subsequent apheresis and manufacturing feasibility for the future EVEREST CEA or MSLN Tmod CAR T-cell studies. Methods BASECAMP-1 (NCT04981119) patient eligibility has 2 parts (figure 2): 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central NGS. If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed for somatic mutations by xTOnco NGS testing; 2) If the tumor demonstrates HLAA* 02:01 LOH and the patient is eligible after screening, the patient will undergo apheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. (Figure Presented)

Background: Metastatic colorectal (CRC), pancreatic (PANC), and gastroesophageal (GE) cancers are the leading causes of GI cancer- related mortality (5-yr survival rate, 14%, 3% and 5-6%, respectively). T-cell immunotherapy targeting GI-associated tumor antigens has been attempted, but efficacy has been constrained by on-target off-tumor toxicity, limiting the therapeutic window. The Tmod (TM) platform is an AND-NOT logic-gated CAR T modular system, versions of which have a CEA-or MSLN-targeting CAR activator and a separate HLA-A*02-targeting blocker receptor to protect normal cells. Tmod CAR T exploits HLA LOH, common in GI malignancies (10-33% in primary solid tumors [TCGA]) and can kill tumor cells without harming healthy cells in vitro and in vivo. However, the prevalence of HLA LOH across GI tumors is unknown in the real-world setting. We utilized the Tempus xT oncology NGS database of patients with multiple GI tumors. From a standard-of-care NGS assay, GI cancer patients can be readily identified for HLA LOH and future treatment with Tmod CAR T therapy.
Method(s): The occurrence of HLA LOH in GI tumors of 1439 patients was assessed using paired germline and somatic DNA sequencing using a research assay [6]. CRC, PANC and GE patients with >= stage 3 were then extracted, and rates of HLA LOH were identified (ie, whether loss occurred across high-frequency HLA-A alleles). In addition, mutations in KRAS and BRAF, as well as MSI status were stratified to determine any association with HLA-A LOH.
Result(s): HLA-A LOH was detected in 830 (17.3%) of all solid tumor records, and a similar proportion when all GI cancer records were analyzed (17.0%). For GI subtypes, these values ranged from 13.5% to 23.1% (Table). No high-frequency HLA-A allele (A*01, A*02, A*03, A*11) was more likely to be lost. Clinical biomarkers (KRAS, BRAF and MSI status) were not associated with HLA-LOH.
Conclusion(s): The frequency of HLA LOH among advanced solid tumor cancers in this dataset is 17.3%, with a range of 13.5-23% between CRC, PANC and GE. The HLA LOH frequency observed in these GI tumors is consistent with that in primary tumors from TCGA, which also used germline-matched and tumor samples. Clinical biomarkers were not associated with HLA LOH. Tempus NGS was able to identify HLA LOH, which can be used for Tmod CAR T therapy to an enhanced therapeutic window. Identification of these patients in BASECAMP-1 (NCT04981119) will enable novel Tmod CAR T therapy. (Table Presented)

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

PURPOSE/OBJECTIVE:To investigate the safety of yttrium-90 radioembolization in combination with checkpoint inhibitor immunotherapy for the treatment of hepatocellular carcinoma (HCC). MATERIALS AND METHODS/METHODS:This single-center retrospective study included 26 consecutive patients with HCC who received checkpoint inhibitor immunotherapy within 90 days of radioembolization from April 2015 to May 2018. Patients had preserved liver function (Child-Pugh scores A-B7) and either advanced HCC due to macrovascular invasion or limited extrahepatic disease (21 patients) or aggressive intermediate stage HCC that resulted in earlier incorporation of systemic immunotherapy (5 patients). Clinical documentation, laboratory results, and imaging results at 1- and 3-month follow-up intervals were reviewed to assess treatment-related adverse events and treatment responses. RESULTS:The median follow-up period after radioembolization was 7.8 months (95% confidence interval [CI], 5.6-11.8). There were no early (30-day) mortality or grades 3/4 hepatobiliary or immunotherapy-related toxicities. Delayed grades 3/4 hepatobiliary toxicities (1-3 months) occurred in 2 patients in the setting of HCC disease progression. One patient developed pneumonitis. The median overall survival from first immunotherapy was 17.2 months (95% CI, 10.9-23.4). The median overall survival from first radioembolization was 16.5 months (95% CI, 6.6-26.4). From first radioembolization, time to tumor progression was 5.7 months (95% CI, 4.2-7.2), and progression-free survival was 5.7 months (95% CI, 4.3-7.1). CONCLUSIONS:Radioembolization combined with checkpoint inhibitor immunotherapy in cases of HCC appears to be safe and causes limited treatment-related toxicity. Future prospective studies are needed to identify the optimal combination treatment protocols and evaluate the efficacy of combination therapy.

Background: Tyrosine kinase inhibitors can prolong survival in advanced HCC patients, but response rates have been minimal. Recently, immune checkpoint inhibition with nivolumab (nivo) demonstrated objective response rates (ORR) of 15% (escalation phase) and 20% (expansion phase) in the Checkmate 040 study. Pre-clinical and translational studies have demonstrated that IL-8 and tumor associated macrophages (TAMs) contribute to HCC progression and recurrence following treatment. Therefore, rationale exists to evaluate combinatorial approaches to target TAM function combined with checkpoint inhibitory therapy. This phase II, randomized study will evaluate the safety and efficacy of combined anti-CSF1R (Cabiralizumab) or anti-IL-8 (BMS-986253) in combination with Nivo in advanced HCC. Method(s): Advanced HCC patients without prior systemic treatment and disease measurable by RECISTv1.1 with Childs A liver function are eligible. Patients will be enrolled (n=25 per arm) to Nivo 240 mg IV Q2 weeks monotherapy, Nivo 240 mg IV + BMS-986253 1200 mg IV Q2 weeks, or Nivo 240 mg IV + Cabiralizumab 4 mg/kg IV Q2 weeks. Primary endpoints include safety and ORR determined by RECISTv1.1. Secondary endpoints include time to response, duration of response, progression free survival, and overall survival. Exploratory endpoints include analysis of tumor microenvironment immune and tumor cell profiling of pre- and on-treatment tumor tissue

Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.